Abstract We have developed a promising leukemia vaccine in which patient derived AML cells are fused with autologous dendritic cells (DCs), presenting a broad array of antigens that capture the heterogeneity of the leukemia cell population. We are completing a phase II clinical trial in which patients that achieve remission following chemotherapy undergo vaccination with DC/AML fusions. Remarkably, 71% of patients with a median age of 63 remain in remission with a median of over four years of follow up. Vaccination was associated with the dramatic expansion of T cells targeting both autologous AML cells and previously defined leukemia associated antigens. We hypothesize that DC/AML vaccination post- allogeneic transplant would elicit the durable expansion of leukemia specific T cells within the donor T cell repertoire to effectively protect against disease relapse. We postulated that vaccine response would be enhanced during donor lymphopoietic reconstitution due to the relative depletion of inhibitory cells and recovery of functionally competent donor cells. Hypomethylating agents (HMAs) have been shown to enhance immunogenicity of tumor cells via enhanced antigen presentation. We have demonstrated that exposure to a second generation HMA is associated with upregulation of antigen processing, increased expression of the PR1 leukemia associated antigen, decreased expansion of MDSCs, and downregulation of PDL1 expression. DC/AML fusions express high levels of PDL-1 offering a counter-regulatory signal that blunts vaccine mediated T cell activation. Notably, antibody mediated blockade of PDL1/PD1 has resulted in durable clinical responses in a subset of patients with advanced malignancies. The efficacy of antibodies targeting the PD-1/PDL-1 pathway as single agents is most pronounced in tumors characterized by a dense infiltrate of tumor infiltrating lymphocytes. In contrast, effective immunotherapy for AML likely requires a strategy to expand leukemia specific lymphocytes and reverse tumor mediated tolerance. Recent studies highlight the importance of additional checkpoint molecules including TIM3 and RGMb that act in concert with PD-1 to induce an exhausted phenotype in tumor reactive lymphocytes. Combined immune checkpoint blockade has the potential to act synergistically to overcome tolerance and enhance immune response to vaccination. In the present study, we will conduct a clinical trial in which AML patients with high risk features who undergo allogeneic transplantation in remission will undergo post- transplant vaccination with donor DC/AML fusions alone or in conjunction with HMA. The primary clinical endpoint is to assess vaccine associated toxicity including the impact on incidence of GVHD. The secondary clinical endpoint will be to examine the effect of vaccination on relapse-free survival. Immunologic response following post-transplant vaccination alone or with HMA will be asssed. In a preclinical murine leukemia model, we will assess the capacity of the second generation checkpoint inhibitors, RGMb and TIM3 alone and in combination with PD-1 blockade to augment response to DC/AML fusion vaccine.